Amino acid substitutions in transmembrane domains 9 and 10 of GerVB that affect the germination properties of Bacillus megaterium spores.
The molecular basis for differences in germinant recognition of Bacillus megaterium QM B1551 spores containing the GerVB and/or GerUB receptor proteins has been examined by site-directed mutagenesis and the construction of cross-homologue chimeras. Focusing on nonconserved residues predicted to reside in transmembrane domains 9 and 10, we demonstrate that GerVB residues Ser319 and Leu345 are of particular importance in defining the specificity and apparent affinity of the receptor for germinants. Kinetic analyses of mutants with different amino acid substitutions at these positions indicate that Ser319 and Leu345 are not involved directly in the binding of germinants, but probably reside in regions of the receptor where structural perturbations can affect the conformation of, or access to, germinant binding sites. Position 345 is also shown to be of importance in GerUB, where the F345A mutation severely impairs receptor function. Functionality is restored in the GerUB Ala345 background by substituting putative outer-loop residues adjacent to TM10 for the corresponding residues in GerVB, indicating that a degree of structural coordination between these regions is important to receptor function.
Christie
Graham
G
Institute of Biotechnology, University of Cambridge, Cambridge, United Kingdom. gc301@cam.ac.uk
Lowe
Christopher R
CR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001410:Bacillus megaterium / Q000235:genetics* / Q000254:growth & development / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids; D011485:Protein Binding; D017422:Sequence Analysis, DNA; D013171:Spores, Bacterial / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D008565:Membrane Proteins; D007930:Leucine; D005947:Glucose